1. Field of the Invention
The present invention relates to novel heterocyclic FRET (fluorescence resonance energy transfer) cassettes, which can be used to label nucleic acids and proteins for a wide variety of molecular biology and DNA sequencing applications.
The currently accepted practice of high throughput gene sequencing employs, as a general rule, four differently labeled Energy Transfer (ET) dye terminators based on the Forster Resonance Energy Transfer (FRET) mechanism to read out the sequence by exciting at one excitation wavelength of the donor and measuring the emissions at the wavelength of the four acceptors conjugated to the four individual nucleic acid bases.
However, the currently available ET terminator sets suffer from low brightness. This low brightness is due to the inefficiencies in the transfer of energy absorbed by the donor to the acceptors and re-emitance at the emission wavelength of the acceptor. This inefficiency arises because of the structural linkages used to join the donor and the acceptors as well as the bases together to form the dye terminators are less than optimal.
The FRET efficiency mainly depends upon the relative dipole-induced-dipole orientation of the participating dyes. The functional groups' orientations with which these dyes are covalently bonded onto the core linker molecule would determine the relative dipole-dipole orientations of the dyes. Heterocyclic and alicyclic molecules with suitable functional groups for making covalent bonds with fluorescent dyes and a molecule of biological interest would serve as cassette cores by virtue of orienting the functional groups in 3D, thereby defining fixed positions for the attached groups. In order to derive highly efficient FRET dye cassettes and turn them into highly sensitive DNA sequencing terminators, heterocyclic systems with different structures and ring sizes were chosen to serve as the core cassette molecules.
In this invention, we offer a novel set of dye labeled cassettes and the corresponding terminators which is brighter than the currently available terminators. The increase in brightness for the set of dye terminators of this invention and the corresponding improvement in signal to noise allow sequencing of a broader range of DNA templates. The novel fluorophore/linker combination, in the form of piperidine or piperazine aminoacid as the core molecule, disclosed in this invention, allows the construction of brighter ET dyes. The heterocyclic FRET cassettes disclosed in this invention can be used to label nucleic acids, proteins, carbohydrates and other biological molecules of interest.
2. Description of Related Art
A large number of fluorescent dyes have been recently developed for labeling and detecting components in biological samples. Generally, these fluorescent dyes must have high extinction coefficient and quantum yield so a low detection limit can be achieved.
One class of dyes which have been developed to give large and different Stokes shifts, based on the Foster Resonance Energy Transfer (FRET) mechanism and used in the simultaneous detection of differently labeled samples in a mixture, are the ET (Energy Transfer) dyes. These ET dyes include a complex molecular structure consisting of a donor fluorophore and an acceptor fluorophore as well as a labeling function to allow their conjugation to biomolecules of interests. Upon excitation of the donor fluorophore, the energy absorbed by the donor is transferred by the Forster Resonance Energy Transfer (FRET) mechanism to the acceptor fluorophore and causes it to fluoresce. Different acceptors can be used with a single donor to form a set of ET dyes so that when the set is excited at one single donor frequency, various emissions can be observed depending on the choice of the acceptors. Upon quantification of these different emissions, the components of a mixture can readily be resolved when these dyes are conjugated to bio-molecules of interest. These ET dye sets constitute the backbone of current high throughput gene sequencing methodology.
Previously, a variety of combinations of bi-fluorophore dyes have been described. U.S. Pat. No. 5,688,648, entitled “Probes Labelled with Energy Transfer Coupled Dyes” Mathies et.al., U.S. Pat. No. 5,728,528, entitled “Universal spacer/energy transfer dyes, and U.S. Pat. No. 6,150,107, entitled “Methods of sequencing and detection using energy transfer labels with cyanine dyes as donor chromophores” which are incorporated herein by reference in its entirety, including any drawings, discloses sets of fluorescent labels carrying pairs of donor and acceptor dye molecules wherein the labels can be attached to nucleic acid backbone for sequencing. The nucleic acid bases or the abasic sugar units are used as spacers to separate the donor and acceptor dyes. The optimum distance for efficient energy transfer from the donor dye to the acceptor dye was found to be ˜6-10 bases. Included is a method for identifying and detecting nucleic acids in a multi-nucleic acid mixture by using different fluorescent labels, wherein the fluorescent moieties are selected from families such as cyanine dyes and xanthenes. The fluorescent labels comprise pairs of fluorophores where one fluorophore donor has emission spectra, which overlaps the fluorophore acceptor's absorption so that there is energy transfer from the excited member to the other member of the pair.
U.S. Pat. No. 6,008,373, entitled “Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer” Waggoner et.al., which is incorporated herein by reference in it's entirety, including any drawings, discloses complexes comprising a first fluorochrome having first absorption and emission spectra and a second fluorochrome having second absorption and emission spectra. The linker groups between the fluorochromes are alkyl chains. The fluorescent nature of the dyes enables them to be of use in sequencing and nucleic acid detection.
U.S. Pat. No. 5,863,727, entitled “Energy transfer dyes with enhanced fluorescence” Lee et al., which is incorporated herein by reference in its entirety, discloses energy transfer dyes in which the donor and acceptor dyes are separated by a linker between the dyes. The preferred linker between the dyes is 4-aminomethylbenzoic acid (Nucleic Acids Research, 1997, 25(14), 2816-2822). The energy transfer terminators DNA sequencing kit based on this linker is commercially available from Applied Biosystems (Foster City, Calif.) and sold as Big Dye terminator kit.
PCT application WO 00/13026 entitled “Energy Transfer Dyes” Kumar et al., which is incorporated herein by reference in its entirety, including any figures and drawings, discloses energy transfer dyes, their preparation, and their use as labels in biological systems. The dyes are preferably in the form of cassettes, which enable their attachment to a variety of biological materials. The donor dye, acceptor dye and the dideoxynucleoside-5′-triphosphates are all attached to a trifunctional linker, which is based on aromatic aminoacids structure (Tetrahedron Letters, 2000, 41, 8867-8871). The energy transfer terminator kit based on these structures is sold by Amersham Biosciences, Piscataway (N.J.) as DYEnamic ET terminator kit for DNA sequencing.
PCT application WO 01/19841 entitled “Charge-modified nucleic acids terminators” Kumar et al., which is incorporated herein by reference in its entirety, including any figures and drawings, discloses single and energy transfer dye labeled terminators with positive or negative charge(s) incorporated in the linker arm. These terminators are useful in generating DNA sequencing bands free of any ‘dye blobs’ which are formed by the degradation of dye labeled dideoxynucleoside-5′-triphosphates. The use of charge terminators allows these degradation products to move backward (positive charge terminators) or move ahead of sequence information (negative charge terminators, Finn et.al. Nucleic Acids Research, 2002, 30(13), 2877-2885).
The currently available ET dye terminator sets, generally, suffer from low brightness. This low brightness is due to the inefficiencies in the transfer of the energy absorbed by the donor to the acceptors and re-emission at the emission wavelength of the acceptor. This inefficiency arises because, the structural linkages used to join the donor, the acceptors, and the nucleic acid bases together to form the dye terminators are less than optimal. Therefore, there remains a need for additional improvements in energy transfer dye cassette construction for maximum brightness and attachment to biological molecules.